Nov 19, 2025

SeV Titration in LLCMK2 Cells V.1

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzjxjxlx1/v1
  • 1Washington University
Carolina Lopez
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzjxjxlx1/v1
Protocol Citation: Carolina Lopez 2025. SeV Titration in LLCMK2 Cells . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjxjxlx1/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 23, 2024
Last Modified: November 19, 2025
Protocol  Integer ID: 100398
Keywords: sev titration in llcmk2 cell, sev titration, llcmk2 cell, protocol for titration, titration, sev
Abstract
Protocol for titration of SeV
Materials


Day 0
The day before titration prepare 96 well plates with LLCMK2 cells. Seed 2x104 cells in 100 µL of Tissue Culture Media (10% fetal calf serum, heat inactivated, endotoxin level 0.25 EU/mL; Hyclone, Logan, UT), 1 millimolar (mM) sodium pyruvate, 2 millimolar (mM) L-glutamine, and 50 mg/mL gentamicin) per well.  
  • Dilute 2x106 cells/plate in 10 mL TCM. Dilute cells to proper concentration then put cell mix in tray and use multichannel to seed cells. Add 100 µL of cell mix/well.
Day 1
3d 1h
1. The day of the titration the cells should be 80-90% confluent.

2. Prepare 1/10 dilutions of the virus in triplicates in Infection Media (DMEM + 50 mg/mL gentamicin + 35% BSA (Immuno, MP Biomedicals) + 5% NaHCO3).
  • Use a different 96 well plate (“dilution plate”)
  • Add 180 µL Infection Media to each well.
  • Add 20 µL virus to first well. Move 20 µL from each well down, changing tips every time.
  • Use a final volume of 200 μL/well for the dilutions. Take out 20 μL of last well.
  • CHANGE TIPS IN EVERY DILUTION.

3. Dump the media of the plate containing the cells and WASH the FBS from the cells by adding/eliminating PBS to the wells. Repeat 2 times. Be careful not to scratch the monolayer.
4. Add 25 µL from the virus dilutions to each well
  • Go from highest dilution to lowest dilution.

5. Incubate for 01:00:00 at 37 °C , 7% CO2.

6. Add 175 µL of Infection Media containing 2 μg/mL TPCK TRYPSIN (Worthington Biochemicals) per well (MAKE SURE YOU DON’T TOUCH THE WELLS WITH THE TIPS).
  • To make 2 μg/mL: 10 mL Infection Media + 10 μL TPCK.

7. Incubate 72:00:00 .
3d 1h
Day 4
35m
1. Wash chicken blood.
  • Add 5 mL chicken blood and 45 mL cold PBS and mix gently.
  • Centrifuge 00:05:00 with 5 acceleration/5 deceleration, 1000 rpm.
  • Discard supernatant.
  • Repeat until supernatant is clear.

OBS.: If using 5% Ready-to-use RBCs (Chicken Red Blood Cells Packed 5%, Innovative Research, Cat. ICHRBC5P15ML), usually one wash is enough since the RBCs come already washed and diluted to 5%.

  • The resulting concentration of chicken RBCs prepared this way is 0.5%.

2. To determine virus titer by hemagglutination of chicken RBCs- to a round bottom 96 well plate add:
  • 25 µL PBS
  • 25 µL media from each well of infected plate.
  • 50 µL chicken blood

3. Agitate plate to mix.

4. Leave plate undisturbed in hood at RT for at least 00:30:00 to allow hemagglutination to occur

5. Score the titer by determining the last dilution with a positive HA. From the triplicates calculate the Tissue Infectious Dose50 / 25 μL.

Note
X = row in which last hemagglutination occurs
+ = no red dot (presence of virus)


Use spreadsheet to calculate titers and HA to infectivity ratios.
35m
Protocol references
Modified for readability by Emmanuelle